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Freeze-dried novel coronavirus fluorescent PCR detection kit and preparation method thereof

A detection kit, coronavirus technology, applied in biochemical equipment and methods, microorganism-based methods, and microbial determination/inspection, etc., can solve problems such as high requirements for experiments and skills, low detection sensitivity, false positives, etc. Good reproducibility, high specificity and sensitivity, ensuring accurate results

Pending Publication Date: 2021-05-14
南通海关综合技术中心
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In the existing technology, the nucleic acid detection reagents that have been listed or patented are all liquid detection reagents, which need to be prepared for immediate use, and have relatively high requirements for experiments and skills; the kits need to be transported in a cold chain, which increases the cost of detection
The detection sensitivity of the colloidal gold detection reagents that have been marketed is relatively low, and there is a certain risk of false negative and false positive
Other published novel coronavirus nucleic acid detection technologies such as Crispr technology [Feng Zhang, Omar O Abudayyeh, Jonathan S Gootenberg. A protocol for detection of COVID-19 using CRISPR diagnosis.], RT-LAMP technology [Lin Yu, Shanshan Wu, Xiaowen Hao, Xuelong Li, Xiyang Liu, ShenglongYe, HengHan, XueDong, XinLi, JiyaoLi, JianminLiu, NaLiu, WanzhongZhang, VicentPelecha no, Wei-HuaChen, XiushanYin. Rapid colorimetric detection of COVID-19 coronavirus using a reverse transcriptional loop mal-mediated isother amplification (RT-LAMP) diagnostic platform: iLACO.doi: https: / / doi.org / 10.1101 / 2020.02.20.20025874] is not as good as RT-qPCR detection reagents in detection throughput and automation

Method used

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  • Freeze-dried novel coronavirus fluorescent PCR detection kit and preparation method thereof
  • Freeze-dried novel coronavirus fluorescent PCR detection kit and preparation method thereof
  • Freeze-dried novel coronavirus fluorescent PCR detection kit and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0085] Example 1: Preparation of freeze-dried novel coronavirus fluorescent PCR detection kit:

[0086] (1) Design and screening of primers and probes

[0087] The novel coronavirus genome sequence downloaded from the NCBI and GSAID databases was compared with the MEGA6.0 biological software, and the conservation of the ORF1ab and N genes announced by the Chinese Center for Disease Control and Prevention (CDC) and the World Health Organization (WHO) were selected. In the region, primers and probes were designed using PrimerExpress2.0 and primer premier5.0 software. The primers and probes were synthesized at Sangon Bioengineering (Shanghai) Co., Ltd. The sequence information of all designed primers and probes is shown in Table 1.

[0088] Table 1 Primer and probe target gene and sequence information

[0089] Table 1 Primer and probe target genes and sequence information

[0090]

[0091] Preparation of 25μl reaction system: 5μl of 60mMTris-HCl, 2.5μl of 10×solution I, 0.5μ...

Embodiment 2

[0104] Embodiment 2: the performance research of kit:

[0105] (1) Sensitivity

[0106] SARS-CoV-2 pseudovirus standard substance (1.0 × 10 4 copies / ml, 1.0×10 3 copies / ml, 5.0×10 2 copies / ml, 2.0×10 2 copies / ml and 1.0×10 2 copies / ml) and negative control were used as test samples for RNA extraction, and the detection sensitivity of the SARS-CoV-2 RT-qPCR freeze-dried detection kit was tested. Each concentration was repeated 20 times, and the detection rate was 95%. 19 positive results were detected as the detection sensitivity of the kit.

[0107] When the sample concentration is greater than 5.0×10 2 When copies / ml, the detection rate of the reagent to positive samples is 100%; when the sample concentration is 2.0×10 2 When copies / ml, the detection rate of ORF1ab gene is 95%, the detection rate of N gene is 100%, that is, the detection rate of SARS-CoV-2 is 95%; for 1.0×10 2 The pseudovirus RNA detection rate of copies / ml is lower than 95% (see Table 2). It shows t...

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Abstract

The invention relates to a freeze-drying type novel coronavirus fluorescent PCR detection kit and a preparation method thereof. The kit comprises an SARS-CoV-2 freeze-drying detection reagent and a quality control reagent, the SARS-CoV-2 freeze-drying detection reagent is an SARS-CoV-2 freeze-drying reaction tube (PCR tube) filled with an SARS-CoV-2 reaction solution, and the main components in the SARS-CoV-2 freeze-drying reaction tube comprise (1) primers (and) probes for detecting the SARS-CoV-2: (1) an ORF1ab gene primer (and) probe, an N gene primer (and) probe and a human beta-actin gene primer (and) probe; (2) an enzyme mixture: TaqDNA polymerase, reverse transcriptase and an RNA enzyme inhibitor; (3) PCR reaction Buffer: Tris-HCl, dNTP, Mg<2+> and the like; and (4) a freeze-drying protective agent. The novel coronavirus nucleic acid detection kit disclosed by the invention has the advantages of high sensitivity, good specificity, normal-temperature transportation, convenience in use and the like.

Description

technical field [0001] The invention relates to the technical field of molecular biological detection of viruses, in particular to a freeze-dried novel coronavirus fluorescent PCR detection kit and a preparation method. Background technique [0002] The 2019-nCoV belongs to the genus β-coronavirus, which has an envelope, and the particles are round or oval, with a diameter of 60nm to 140nm. The main clinical manifestations are fever, dry cough, and fatigue. Severe patients gradually develop more serious diseases such as dyspnea and / or hypoxemia; severe patients rapidly progress to acute respiratory distress syndrome, septic shock, and metabolic acidity that is difficult to correct. Poisoning and coagulation disorders [Huang C, Wang Y, Li X, Ren L, Zhao J, Hu Y, Zhang L, Fan G, Xu J, Gu X, Cheng Z, Yu T, Xia J, Wei Y, Wu W , Xie X, Yin W, Li H, Liu M, Xiao Y, Gao H, Guo L, Xie J, Wang G, Jiang R, Gao Z, JinQ, Wang J, Cao B. Clinical features of patients infected with 2019 no...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/686C12N15/11C12R1/93
CPCC12Q1/701C12Q1/686C12Q2600/166C12Q2600/16C12Q2563/107C12Q2537/143
Inventor 陈峰吴海磊唐晓宇张阳刘文干
Owner 南通海关综合技术中心
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